Project description:Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neuron. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. Overall design: DEG analysis of primary OPC and OL populations, 5 biological replicates per population
Project description:Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a pro-inflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from primary progressive multiple sclerosis (PPMS) patient induced pluripotent stem (iPS) cell lines failed to promote oligodendrocyte progenitor cell (OPC) maturation whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+ progenitor cells within white matter lesions of human progressive MS autopsy brain tissues and PPMS patient iPS-derived NPCs. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin which then enhanced PPMS NPC support for oligodendrocyte differentiation. A proteomic analysis of the PPMS NPC secretome identified high mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of oligodendrocyte differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in progressive MS which may impact how this disease is modeled and inform development of future myelin regeneration strategies.
Project description:The zinc finger protein ZFP24 is critical for CNS myelination. Nonetheless, the mechanism by which ZFP24 controls myelination is unknown. Here we use chromatin IP (ChIP) to map ZFP24 binding sites in oligodendrocyte progenitor cells (OPC) and differentiated oligodendrocytes (OLG). We find that ZFP24 directly binds the enhancer regions of genes important for oligodendrocyte differentiation and myelination and mediates their expression. We demonstrate that ZFP24 undergoes phosphorylation and dephosphorylation in oligodendrocyte lineage cells and that ZFP24 binding to DNA is controlled by its phosphorylated state such that only the non-phosphorylated form of the protein, predominantly found in mature oligodendrocytes, mediates expression of myelin protein genes. We have also identified key ZFP24 downstream target genes. Among these, we show that enforced expression of the crucial myelin transcription factor MYRF can rescue myelin proteins gene expression in ZFP24 -ablated cells. Our data also suggest that ZFP24 display overlapping genomic binding sites with the transcription factors MYRF, SOX10, and OLIG2 which are known to control terminal differentiation of oligodendrocytes. Though the human genome contains roughly 700 C2H2-containing zinc finger proteins, the DNA-binding sequences and the biological functions of the vast majority of them are unknown. Our findings provide a direct molecular mechanism by which dephosphorylation of ZFP24 mediates its binding to enhancer regions of genes important for oligodendrocyte differentiation and myelination, controls their expression, and as a result, regulates oligodendrocyte differentiation and CNS myelination. Overall design: Chromatin IP for ZFP24 was performed on mouse Oligodendrocyte progenitor cells (OPC) and mature (differentiated) oligodendrocyte (OLG). Corresponding Zfp24 null cells were used as control, as well as pooled chromatin input.
Project description:To quantitative analysis of transcriptome changes caused by lnc-OPC knockdown during OPC differentiation from NSC, lentivirus-based short hairpin RNAs were used to knockdown the lnc-OPC expression in a neural stem cell culture . Subsequently, puromycin-selected NSCs were differentiated to OPC in culture for three days.RNA-Seq was performed on the polyadenylated fraction of RNA isolated from cell samples. DEseq was used for differential gene expression analysis caused by lnc-OPC knockdown. GO functional term enrichment analysis of differential gene expression caused by lnc-OPC knockdown, revealed significant enrichment of ‘oligodendrocyte development’, ‘oligodendrocyte differentiation’, ‘glia cell development’, and ‘axon ensheathment’ terms that are associated with oligodendrogenesis. mRNA profiles of differentiiated NSC samples after lnc-OPC knockdown by RNA-sequencing.
Project description:Vascular pericytes, an important cellular component, in the tumor microenvironment, are often associated with tumor vasculatures and their functions in cancer invasion and metastasis are poorly understood. Here we show that PDGF-BB induces pericyte fibroblast transition (designated as PFT), which significantly contributes to tumor invasion and metastasis. Gain- and loss-of-function experiments demonstrate that the PDGF-BB-PDGFRβ signaling promotes PFT in vitro and in in vivo tumors. Genome-wide expression analysis indicates that PDGF-BB-activated pericytes acquire mesenchymal progenitor features. Pharmacological inhibition and genetic deletion of PDGFRβ ablate the PDGF-BB-induced PFT. Genetic tracing of pericytes with two independent mouse strains, i.e., TN-AP-CreERT2:R26R-tdTomato and NG2:R26R-tdTomato, shows that PFT cells gains stromal fibroblast and myofibroblast markers in tumors. Importantly, co-implantation of PFT cells with less-invasive tumor cells in mice markedly promotes tumor dissemination and invasion, leading to an increased number of circulating tumor cells (CTCs) and metastasis. Our findings reveal a novel mechanism of vascular pericytes in PDGF-BB-promoted cancer invasion and metastasis by inducing PFT and thus targeting PFT may offer a new treatment option of cancer metastasis. Pericytes were isolated and treated with PDGF-BB or control for 1 or 5 days
Project description:The mechanisms underlying the specification of oligodendrocyte fate from multipotent neural progenitor cells (NPCs) in developing human brain are unknown. In this study, we sought to identify antigens sufficient to distinguish NPCs free from oligodendrocyte progenitor cells (OPCs). We investigated the potential overlap of NPC and OPC antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFaR antigens. Surprisingly, we found that CD133, but not A2B5, was capable of enriching for OLIG2 expression, Sox10 enhancer activity, and oligodendrocyte potential. As a subpopulation of CD133- positive cells expressed CD140a, we asked whether CD133 enriched bone fide NPCs regardless of CD140a expression. We found that CD133+CD140a- cells were highly enriched for neurosphere initiating cells and were multipotent. Importantly, when analyzed immediately following isolation, CD133+CD140a- NPCs lacked the capacity to generate oligodendrocytes. In contrast, CD133+CD140a+ cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Gene expression analysis further confirmed the stem cell nature of CD133+CD140a- cells. As human CD133+ cells comprised both NPCs and OPCs, CD133 expression alone cannot be considered a specific marker of the stem cell phenotype, but rather comprises a heterogeneous mix of glial restricted as well as multipotent neural precursors. In contrast, CD133/CD140a-based FACS permits the separation of defined progenitor populations and the study of neural stem and oligodendrocyte fate specification in the human brain. 12 samples, 4 groups (FACS-sorted cell populations),3 replicates in each group, each replicate is from a separate patient sample
Project description:PRMT5 is important for gliomas, however its physiological function in oligodendrocyte progenitors (OPCs), remains poorly understood. Here we report PRMT5 to be responsible for symmetric di-methylation of histone H4R3 (H4R3me2s) in OPC. PRMT5 depletion via CRISPR/Cas9 decreased H4R3me2s, altered the OPC transcriptome, and decreased OPC survival. Strikingly, these changes were associated with a marked increase of H4K5ac in OPC, but not in gliomas. Consistently, ChIP-sequencing analysis revealed increased genome-wide distribution of H4K5ac in PRMT5 knockout cells. In vitro acetylation assays demonstrated reciprocal inhibition between symmetric arginine methylation and lysine acetylation. Low H4R3me2s and high H4K5ac levels were also detected in OPC in mice with lineage-specific ablation of Prmt5 (Olig1-Cre;Prmt5fl/fl), resulting in severely impaired developmental myelination. Importantly, pharmacological inhibition of acetyltransferase activity partially rescued the effect of Prmt5 deletion on oligodendrocyte-specific gene transcripts. Collectively, we identify PRMT5 as a critical modulator of histone acetylation and OPC differentiation in early developmental myelination. Overall design: (1) mRNA profiles of OPCs with or without PRMT5 expression were generated by deep sequencing, in tetraplicates, using Illumina HiSeq 2500.
Project description:Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to ‘induced’ oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphologyical and global gene expression profile molecular features consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into induced multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelinmyelinating axons both in vitro and in vivo. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies. 6 total samples were analyzed. MEFs were either untreated or infected with inducible lentiviral vectors containing the open reading frames of transcription factors. Samples were compared to bona fide OPCs.
Project description:The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB. The brain microvascular fragments were isolated from mice with different genotypes, each represented by 3-4 biological replicates. Genotypes 1-2: Platelet derived growth factor-B (PDGF-B) retention-motif knockout (pdgfbret/ret) represent the pericyte-deficient situation, and the heterozygous mice (pdgfbret/+) are used as controls. Genotypes 3-4: Hypomorphic PDGF-B mutants that rescue pdgfb-/- null mice, in which a one copy of a conditionally silent human PDGF-B transgene targeted to the Rosa 26 locus (R26P) is turned on by endothelial-specific expression of Cre recombinase. In this data set these mice are named as Tie2Cre, R26P+/0, pdgfb-/- (representing the pericyte-deficient situation). Mice wt for pdgfb (pdgfb+/+) and carrying one silent copy of R26P (R26P+/0), are used as controls. Genotype 5: Adult Notch3+/+ wildtype (WT).
Project description:Actin assembly and dynamics control the shape, motility and functions of diverse cell types. Programme for controlling theses dynamics is hard-coded into actin binding proteins and regulatory proteins. Refilins (RefilinA and RefilinB) are short lived regulatory proteins that interact with the actin-binding protein Filamin to convert it from an actin branching protein into one that bundles. We here show that different mechanisms converge to increase Refilin level in brain NG2+ precursor cells (polydendrocytes) committed to differentiate into the oligodendrocyte lineage. RefilinA is up-regulated through transcriptional activation during commitment into oligodendrocyte progenitor cells (OPC). RefilinB expression relies on PDGF signalling and is further stabilized by a unique auto-inhibitory domain masking the adjacent conserved PEST degradation signal. In NG2+ precursor cells and OPC, the RefilinB/Filamin complex localizes on filipodia protrusions and filipodial processes. Stable ectopic expression of Refilins in cells stimulates membrane remodelling dynamics linked with filipodia growth. These studies extend the function of the Refilin/FLNA complex to cell membrane remodelling linked with reorganization of underlying actin cytoskeleton. We performed whole transcriptomic analysis on the three cell population derived from OPC cells. Three different cultures were done and for each experiment we compared each cell population (NG2+, NG2+/A2B5 and O4) to the other 2 cell populations.